Eukaryotic gene expression must be regulated such that cells can rapidly respond to a wide range of different conditions. The process of mRNA translation is one step at which gene expression is highly regulated. In response to hormones, growth factors, cytokines and nutrients, animal cells generally activate translation in preparation for the proliferative response. The rate of protein synthesis typically decreases under stressful conditions, such as oxidative or osmotic stress, DNA damage or nutrient withdrawal. Activation or suppression of mRNA translation occurs within minutes and control over this process is thought to be exerted at the initiation phase of protein synthesis (Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Strudwick and Borden, Differentiation, 2002, 70, 10-22).
Translation initiation necessitates the coordinated activities of several eukaryotic initiation factors (eIFs), proteins which are classically defined by their cytoplasmic location and ability to regulate the initiation phase of protein synthesis. One of these factors, eukaryotic initiation factor 4E (eIF4E) (also known as eukaryotic translation initiation factor 4E, eukaryotic translation initiation factor 4E-like 1 (eIF4EL 1), cap-binding protein (CBP) and messenger RNA cap-binding protein) was initially isolated as a 25 kDa mRNA cap-binding protein involved in translation (Rychlik et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 945-949) and has since become one of the most highly-characterized eIFs. eIF4E, present in limiting amounts relative to other initiation factors, is one component of the eIF4F initiation complex, which is also comprised of a scaffold protein eIF4G and the RNA helicase eIF4A. In the cytoplasm, eIF4E catalyzes the rate-limiting step of cap-dependent protein synthesis by specifically binding to the 5′ terminal 7-methyl GpppX cap structure present on nearly all mature cellular mRNAs, which serves to deliver the mRNAs to the eIF4F complex. Once bound, the eIF4F complex scans from the 5′ to the 3′ end of the cap, permitting the RNA helicase activity of eIF4A to resolve any secondary structure present in the 5′ untranslated region (UTR), thus revealing the translation initiation codon and facilitating ribosome loading onto the mRNA (Graff et al., Clin. Exp. Metastasis, 2003, 20, 265-273; Strudwick et al., Differentiation, 2002, 70, 10-22).
eIF4E availability for incorporation into the eIF4E complex is regulated through phosphorylation as well as through the binding of inhibitory proteins. eIF4E is a phosphoprotein that is phosphorylated on serine 209 by the mitogen-activated protein kinase-interacting kinase Mnk1 (Flynn et al., J. Biol. Chem., 1995, 270, 21684-21688; Wang et al., J. Biol. Chem., 1998, 273, 9373-9377; and Waskiewicz et al., Embo J., 1997, 16, 1909-1920). Phosphorylation of eIF4E increases its affinity for mRNA caps, thus elevating translation rates (Waskiewicz et al., Mol. Cell. Biol., 1999, 19, 1871-1880). Increased phosphorylation of eIF4E by phorbol esters, cell stresses and cytokines involves the p38 mitogen-activated (MAP) kinase and/or Erk signaling pathways, which in turn stimulate Mnk1 activity. Other stresses such as heat shock, sorbitol and hydrogen peroxide stimulate p38 MAP kinase and increase Mnk1 activity, however, these stimuli increase the binding of eIF4E to the eIF4E-binding protein 1 (4E-BP1) (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377). Binding of 4E-BP1 to eIF4E blocks the phosphorylation of eIF4E by Mnk1 (Wang et al., J. Biol. Chem., 1998, 273, 9373-9377). The 4E-binding proteins 1 and 2 act as effective inhibitors of translation by competing with eIF4G for binding to the dorsal surface of eIF4E (Ptushkina et al., Embo J., 1999, 18, 4068-4075). Phosphorylation of the binding proteins by MTOR causes them to dissociate from eIF4E, allowing eIF4E activity.
A growing number of observations suggest that translation factors localize and function in the nucleus, as well as in the cytoplasm. Transcription and translation are traditionally considered to be spatially separated in eukaryotes; however, coupled transcription and translation is observed within the nuclei of mammalian cells (Iborra et al., Science, 2001, 293, 1139-1142). A fraction of eIF4E localizes to the nucleus, suggesting that this translation factor may exhibit some of its control over translation in the nucleus (Lejbkowicz et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 9612-9616). eIF4E is imported into the nucleus through the importin alpha/beta pathway by the nucleoplasmic shuttling protein eIF4E-transporter (4E-T) (Dostie et al., Embo J., 2000, 19, 3142-3156). In the nucleus, eIF4E can be directly bound by the promyelocytic leukemia protein (PML), an important regulator of mammalian cell growth and apoptosis (Cohen et al., Embo J., 2001, 20, 4547-4559). PML, through its RING domain, modulates eIF4E activity by greatly reducing its affinity for the 5′ cap structure of mRNAs (Cohen et al., Embo J., 2001, 20, 4547-4559).
An excess of eIF4E does not lead to global elevated translation rates, but rather selectively increases the synthesis of proteins encoded by mRNAs that are classified as eIF4E-sensitive, including growth stimulatory proteins such as vascular endothelial growth factor (VEGF), ornithine decarboxylase (ODC) and cyclin D1 (Kevil et al., Int. J. Cancer, 1996, 65, 785-790; Rosenwald, Cancer Lett., 1995, 98, 77-82; and Shantz et al., Cancer Res., 1994, 54, 2313-2316). While ODC and VEGF protein levels are elevated through increased translation initiation, cyclin D1 levels are elevated due to greater transport of cyclin D1 mRNA into the cytoplasm (Kevil et al., Int. J. Cancer, 1996, 65, 785-790; Rosenwald, Cancer Lett., 1995, 98, 77-82; Rousseau et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 1065-1070). Thus, in addition to having a role in translation initiation, eIF4E can also affect mRNA nucleocytoplasmic transport.
eIF4E function is an essential determinant of overall cell protein synthesis and growth (De Benedetti et al., Mol. Cell. Biol., 1991, 11, 5435-5445). In normal cells, eIF4E is present in limiting amounts, which restricts translation. mRNAs which encode proteins necessary for cell growth and survival typically contain a complex, highly structured 5′ UTR, which renders these mRNAs poor substrates for translation. Many of these mRNAs, however, are well translated in the presence of excess eIF4E and are also upregulated by tumors (Graff and Zimmer, Clin. Exp. Metastasis, 2003, 20, 265-273). The translation of mRNAs related to cell differentiation may also be enhanced by eIF4E, as increased levels of eIF4E are found in some differentiating cell lines, including epithelial lung tumor cell lines (Walsh et al., Differentiation, 2003, 71, 126-134).
Overexpression of eIF4E has been reported in many human cancers and cancer-derived cell lines and also leads to oncogenic transformation of cells and invasive/metastatic phenotype in animal models. Unlike non-transformed, cultured cells, transformed cell lines express eIF4E independently of the presence of serum growth factors (Rosenwald, Cancer Lett., 1995, 98, 77-82). Excess eIF4E leads to aberrant growth and neoplastic morphology in HeLa cells and also causes tumorigenic transformation in NIH 3T3 and Rat2 fibroblasts, as judged by anchorage-independent growth, formation of transformed foci in culture and tumor formation in nude mice (De Benedetti et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 8212-8216; and Lazaris-Karatzas et al., Nature, 1990, 345, 544-547). Furthermore, neoplastic transformation exhibited by cells overexpressing eIF4E is associated with the increased translation of ODC (Lazaris-Karatzas et al., Nature, 1990, 345, 544-547). Additionally, the elevated nuclear export of cyclin D1 associated with increased eIF4E expression is directly linked to transformation activity (Cohen et al., Embo J., 2001, 20, 45474559). These findings demonstrate that when present in excess, eIF4E can increase the expression or nuclear export of growth regulatory mRNAs. As a consequence, the affected cells can proliferate independently of normal growth control mechanisms. Enhanced eIF4E phosphorylation is observed in cells transformed with the src tyrosine kinase oncoprotein, suggesting that elevated eIF4E activity, in addition to overexpression, contributes to the loss of growth regulation in transformed cells (Frederickson et al., Mol. Cell. Biol., 1991, 11, 2896-2900).
eIF4E is found elevated in several human cancers, including but not limited to non-Hodgkin's lymphomas, colon adenomas and carcinomas and larynx, head and neck, prostate, breast and bladder cancers (Crew et al., Br. J. Cancer, 2000, 82, 161-166; Graff et al., Clin. Exp. Metastasis, 2003, 20, 265-273; Haydon et al., Cancer, 2000, 88, 2803-2810; Kerekatte et al., Int. J. Cancer, 1995, 64, 27-31; Rosenwald et al., Oncogene, 1999, 18, 2507-2517; Wang et al., Am. J. Pathol., 1999, 155, 247-255). Upregulation of eIF4E is an early event in colon carcinogenesis, and is frequently accompanied by an increase in cyclin D1 levels (Rosenwald et al., Oncogene, 1999, 18, 2507-2517). Excess eIF4E is also a reliable predictor of tumor recurrence in head and neck carcinomas, is selectively upregulated in invasive bladder carcinomas and is correlated with poor histological grades and more advanced states of metastasis in laryngeal squamous cell carcinoma (Crew et al., Br. J. Cancer, 2000, 82, 161-166; Liang et al., Laryngoscope, 2003, 113, 1238-1243; and Nathan et al., Oncogene, 1997, 15, 579-584). These findings suggest that elevated levels of eIF4E participate in the advancement as well as initiation of cancer.
Inhibition of eIF4E expression and activity has been accomplished through the use of antisense mechanisms. Antisense oligonucleotides equipped with 3′-overhanging nucleotides modulate the binding of eIF4E to 5′-capped oligoribonucleotides (Baker et al., J. Biol. Chem., 1992, 267, 11495-11499). Introduction into HeLa cells of an episomal vector engineered to express an oligonucleotide complementary to 20 nucleotides in the translation start region of eIF4E reduces levels of eIF4E and concomitantly decreases the rates of cell growth and protein synthesis, demonstrating that eIF4E is required for cell proliferation (Bommer et al., Cell. Mol. Biol. Res., 1994, 40, 633-641; De Benedetti et al., Mol. Cell. Biol., 1991, 11, 5435-5445). Levels of eIF4G, the scaffold protein component of the eIF4F complex, are also reduced. Despite the diminished levels of translation following inhibition of eIF4E, certain proteins continue to be synthesized, and many of these have been identified as stress-inducible or heat-shock proteins (Joshi-Barve et al., J. Biol. Chem., 1992, 267, 21038-21043). The same vector reduces eIF4E by 50 to 60 percent in rat embryo fibroblasts, which is sufficient to inhibit ras-mediated transformation and tumorigenesis of these cells (Graff et al., Int. J. Cancer, 1995, 60, 255-263; Rinker-Schaeffer et al., Int. J. Cancer, 1993, 55, 841-847). Furthermore, ODC translation and polyamine transport are diminished, an observation that provides a link between ras-induced malignancy, eIF4E activity and polyamine metabolism (Graff et al., Biochem. Biophys. Res. Commun., 1997, 240, 15-20). Stable transformation of a mammary carcinoma line and a head and neck squamous cell carcinoma cell line with the eIF4E antisense vector results in reduction fibroblast growth factor-2 (FGF-2) expression and in inhibition of turnorigenic and angiogenic capacity of the cells in mice, suggesting a causal role for eIF4E in tumor vascularization (DeFatta et al., Laryngoscope, 2000, 110, 928-933; Nathan et al., Oncogene, 1997, 15, 1087-1094).
Targeted inactivation of a Caenorhabditis elegans homolog of human eIF4E, IFE-3, with small interfering RNA injected into young adult worms leads to embryonic lethality in 100% of the progeny (Keiper et al., J. Biol. Chem., 2000, 275, 10590-10596). Small interfering double-stranded RNA targeted to eIF4E has also revealed that lack of eIF4E regulation participates in cellular transformation. Functional inactivation of eIF4E using a gene-specific 21-nucleotide small interfering RNA targeted to a portion of the coding region of human eIF4E results in a significant reduction of anchorage-independent growth of malignant cholangiocytes, a phenotype associated with transformed cells. In addition, phosphorylation of eIF4E in malignant cholangiocytes is dependent upon p38 MAP kinase signaling, demonstrating a link between p38 MAP kinase signaling and the regulation of protein synthesis in the process of cholangiocarcinoma growth (Yamagiwa et al., Hepatology, 2003, 38, 158-166).
Further evidence that inhibition of eIF4E activity reduces the tumorigenic potential of cells is seen in breast cancer cells that express a constitutively active form of the eIF4E inhibitor 4EBP-1, which leads to cell cycle arrest associated with downregulation of cyclin D1 and upregulation of the cyclin-dependent kinase p27Kip1 (Jiang et al., Cancer Cell Int., 2003, 3, 2). The overexpression of 4E-BP1 in gastrointestinal cancers, where eIF4E levels are significantly higher than in normal tissue, is correlated with a reduction in distant metastases (Martin et al., Int. J. Biochem. Cell. Biol., 2000, 32, 633-642).
U.S. Pat. No. 5,646,009 claims and discloses a hybrid vector in which one DNA segment encodes a cap-binding protein consisting of eIF4E, eIF4E factor or a mutant thereof. This patent also discloses a nucleic acid sequence encoding a human eIF4E.
Disclosed in U.S. Pat. No. 6,171,798 is a method for treating cancer in a patient by administering to cancer cells an antisense construct comprising at least 12 nucleotides of a coding sequence of a gene selected from a group containing a human eIF4E, in 3′ to 5′ orientation with respect to a promotor controlling its expression.
U.S. Pat. No. 6,596,854 claims and discloses isolated nucleic acid molecules encoding variants of human eIF4E, wherein said variants have amino acid substitutions in the regions of amino acids 112 and 114-121, or position 118, or position 119, or position 115 or position 121.
European patent application 1 033 401 and Japanese patent application 2001269182 claim a purified nucleic acid comprising at least 10 consecutive nucleotides of a sequence selected from a group of EST-related sequences which includes a portion of a nucleic acid molecule encoding human eIF4E. These publications also disclose the preparation and use of antisense constructs and oligonucleotides to be used in gene therapy.
PCT publications WO 01/96388 and WO 01/96389 disclose and claim isolated polynucleotides comprising a sequence selected from: sequences, complements of sequences, sequences consisting of at least 20 contiguous residues of a sequence, sequences that hybridize to a sequence, or sequences having at least 75% or at least 95% identity to a sequence, provided in the sequence listing, which includes a nucleic acid molecule encoding a human eIF4E. This publication also claims a method for the treatment of a cancer in a patient, comprising administering to the patient a composition of the claimed polynucleotides.
PCT publication WO 03/039443 claims aid discloses a method for the preparation of a pharmaceutical composition for the treatment of leukemia characterized in that an antisense oligonucleotide complementary to a polynucleotide encoding a protein corresponding to marker, selected from a group including a human eIF4E nucleic acid molecule, is admixed with pharmaceutical compounds.
U.S. pre-grant publication 20030087852 discloses a plasmid encoding eIF4E antisense mRNA and cultured mouse cells transfected with this plasmid.
Disclosed in U.S. pre-grant publication 20030144190 are antisense molecules which may be used to decrease or abrogate the expression of a nucleic acid sequence or protein of the invention, including eIF4E. Also disclosed are a plasmid encoding eIF4E antisense mRNA and cultured rat fibroblasts constitutively expressing this plasmid.
As a consequence of eIF4E involvement in many diseases, there remains a long felt need for additional agents capable of effectively regulating eIF4E. As such, inhibition is especially important in the treatment of cancer, given that the upregulation of expression of eIF4E is associated with so many different types of cancer.
Antisense technology is an effective means for reducing the expression of specific gene products and has been proven to be uniquely useful in a number of therapeutic, diagnostic, and research applications. The present invention provides compositions and methods for modulating eIF4E expression.